From owner-biophys@hgmp.mrc.ac.uk Thu Jul 24 13:54:39 2003 Return-Path: X-Original-To: biophys-outgoing Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with SMTP id 73F997D2EE for ; Thu, 24 Jul 2003 13:54:39 +0100 (BST) X-Original-To: biophys-list@hgmp.mrc.ac.uk Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id CDAAF7D326 for ; Thu, 24 Jul 2003 13:54:38 +0100 (BST) Received: from niobium.hgmp.mrc.ac.uk (niobium [193.62.192.41]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id EC9F07D2EE for ; Thu, 24 Jul 2003 13:54:37 +0100 (BST) Received: (from news@localhost) by niobium.hgmp.mrc.ac.uk (8.11.6+Sun/8.8.8) id h6OCsbg22186 for biophys-list@hgmp.mrc.ac.uk; Thu, 24 Jul 2003 13:54:37 +0100 (BST) X-Authentication-Warning: niobium.hgmp.mrc.ac.uk: news set sender to using -f From: frank@kuesterei.ch (=?iso-8859-15?q?Frank_K=FCster?=) X-Newsgroups: bionet.biophysics Subject: strange baseline behavior of diode array photometer Date: Thu, 24 Jul 2003 14:22:41 +0200 Lines: 52 Message-ID: <874r1ct8q6.fsf@alhambra.bioz.unibas.ch> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii X-Orig-Path: alhambra.bioz.unibas.ch!news X-Attribution: fant X-Ehrenamt: http://www.langau.de User-Agent: Gnus/5.1002 (Gnus v5.10.2) Emacs/21.3 (gnu/linux) Cancel-Lock: sha1:6LHnzeZFifaQzGiuoRSLFWx8eOI= To: biophys@hgmp.mrc.ac.uk Sender: owner-biophys@hgmp.mrc.ac.uk Precedence: bulk Hi all, Yesterday I experienced a strange behavior on a diode array photometer. Although I had scanned a baseline with a buffer blank, the baseline was in part negative. ^ A | | ,`\ 0.3-|/ \ | | | \ | | | \ | | | \ | | 0-| \ ________-------------------- | \ _______------ | `----- +--------------------------------------------------------> 280 320 340 380 420 The buffer was 10 mM Naphosphate, pH 7.0, the sample was a protein with an A_280nm of 0.356 as measured on a different instrument. However, the measured "absorption" of the protein sample was zero only at very high wavelenghts (800 nm). Below that it slowly decreased nonlinearly until it reached a minimum at about 315 or 320 nm - then the protein absorption started and made the signal increase. The value at the minimum was about -0.04. I have the feeling that it was a problem of the instrument, because: - when I put the same sample into an other instrument (a normal monochromator photometer), it had a fine baseline at 0.00 above 310 nm. - I had found the opposite behavior (approximately linear decrease in OD above 310 nm) on that instrument quite often and attributed it to light scattering, but no I am in doubt, because this protein usually doesn't tend to aggregate. Does anybody have an idea how this can come, and what I should do? (There's one other possibility: I had to change the cuvette (Hellma 1cm 104-QS in both cases) when going to the other instrument. But I used it in the same orientation for buffer blank and sample measurement). Thanks in advance, Frank -- sigmentation fault .